Lung cancer is the leading cause of cancer-related death for men and women in the U.S. The susceptibility of the chronic smoker for developing lung cancer is likely linked to germ-line polymorphisms in critical genes involved in the bioactivation, detoxification, and repair of DNA damage stemming from tobacco carcinogens and the subsequent acquisition of somatic genetic and epigenetic changes in critical regulatory genes. Epigenetically mediated gene silencing through promoter hypermethylation is a common event that is critical for initiation and progression of cancer. Population-based studies of cancer-free male and female smokers have not been conducted to evaluate the interrelationships between clinical risk factors, germ-line, and somatic changes for their contribution to lung cancer development. In support of this approach, we have demonstrated that aberrant methylation of the pl6 and/or O6-methylguanine-DNA methyltransferase (MGMT) promoters can be detected in DNA from sputum in 100% of squamous cell lung carcinomas (SCCs) up to 3 years before clinical diagnosis. The extension of these findings to a nested, case-control study revealed that the presence of any one of four methylation markers examined was associated with a 6.3 fold increase in risk for incident lung cancer. In addition, we conducted a pilot study to determine whether germ-line mutations in lung cancer susceptibility genes are associated with the presence of methylated alleles of p 16 and MGMT in sputum from cancer-free, high-risk subjects. Risk alleles for the genes encoding the NAD(P)H quinone oxidoreductase and glutathione-S-transferase P1 genes were significantly associated with methylation of the pl6 or MGMT genes. These findings will be extended to integrate metabolic susceptibility factors with genetic biomarkers and clinical risk factors to better understand the etiology of lung cancer. Four specific aims will be accomplished through a cross-sectional and longitudinal study using 1500 subjects, 750 from each of the two existing cohorts of current and former smokers, a 2,250-person cohort of veterans, and a 1,500-person female cohort. The first will determine whether the risk alleles for genes involved in tobacco carcinogen activation and detoxification, oxidative stress, DNA repair, and de novo methylation of CpG sites are associated with gene-specific promoter hypermethylation in sputum. The second aim will determine whether diet and/or the established clinical risk factors COPD and smoking history are associated with DNA methylation detected in exfoliated cells within sputum. The third aim will determine whether the effect of clinical risk factors that include smoking on methylation patterns vary as a function of genotype. Finally we will describe the gene-specific promoter hypermethylation patterns at study entry and at 18-month follow-up.